Enhancement of Bleomycin Production in Streptomyces Verticillus Through Global Metabolic Regulation of N-Acetylglucosamine and A

Chen et al. Microb Cell Fact (2020) 19:32 https://doi.org/10.1186/s12934-020-01301-8 Microbial Cell Factories RESEARCH Open Access Enhancement of bleomycin production in Streptomyces verticillus through global metabolic regulation of N-acetylglucosamine and assisted metabolic profling analysis Hong Chen1,2†, Jiaqi Cui1,2†, Pan Wang1,2, Xin Wang1,2 and Jianping Wen1,2* Abstract Background: Bleomycin is a broad-spectrum glycopeptide antitumor antibiotic produced by Streptomyces verticillus. Clinically, the mixture of bleomycin A2 and bleomycin B2 is widely used in combination with other drugs for the treatment of various cancers. As a secondary metabolite, the biosynthesis of bleomycin is precisely controlled by the complex extra-/intracellular regulation mechanisms, it is imperative to investigate the global metabolic and regulatory system involved in bleomycin biosynthesis for increasing bleomycin production. Results: N-acetylglucosamine (GlcNAc), the vital signaling molecule controlling the onset of development and antibiotic synthesis in Streptomyces, was found to increase the yields of bleomycins signifcantly in chemically defned medium. To mine the gene information relevant to GlcNAc metabolism, the DNA sequences of dasR-dasA-dasBCD- nagB and nagKA in S. verticillus were determined by chromosome walking. From the results of Real time fuorescence quantitative PCR (RT-qPCR) and electrophoretic mobility shift assays (EMSAs), the repression of the expression of nagB and nagKA by the global regulator DasR was released under induction with GlcNAc. The relief of blmT expression repression by BlmR was the main reason for increased bleomycin production. DasR, however, could not directly afect the expression of the pathway-specifc repressor BlmR in the bleomycins gene cluster. With at the beginning of bleomycin synthesis, the supply of the specifc precursor GDP-mannose played the key role in bleomycin production. Genetic engineering of the GDP-mannose synthesis pathway indicated that phosphomannose isomerase (ManA) and phosphomannomutase (ManB) were key enzymes for bleomycins synthesis. Here, the blmT, manA and manB co-expression strain OBlmT/ManAB was constructed. Based on GlcNAc regulation and assisted metabolic profling analysis, the yields of bleomycin A2 and B2 were ultimately increased to 61.79 and 36.9 mg/L, respectively. Conclusions: Under GlcNAc induction, the elevated production of bleomycins was mainly associated with the alleviation of the inhibition of BlmT, so blmT and specifc precursor synthesis pathways were genetically engineered for bleomycins production improvement. Combination with subsequent metabolomics analysis not only efectively increased the bleomycin yield, but also extended the utilization of chitin-derived substrates in microbial-based antibiotic production. *Correspondence: [email protected] †Hong Chen and Jiaqi Cui contributed equally to this work 1 Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300072, People’s Republic of China Full list of author information is available at the end of the article © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdo- main/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Chen et al. Microb Cell Fact (2020) 19:32 Page 2 of 17 Keywords: Bleomycin, GlcNAc, DasR, GDP-mannose, Metabolic profling Introduction defciency, which is delivered and sensed by Streptomy- Bleomycin is a member of glycopeptide antibiotics that ces to initiate sporulation and secondary metabolism was frst isolated from the fermentation broth of S. ver- [17]. Terefore, not only does GlcNAc or chitin provide ticillus by Umezawa et al. [1]. Te bleomycin gene cluster a source of carbon and nitrogen for strain growth, but and its structure clearly indicate that a hybrid peptide- the GlcNAc-induced regulation of antibiotic synthesis polyketide skeleton and a disaccharide unit together should be utilized to improve the bleomycin production constitute bleomycins with diferent C-terminal amines in S. verticillus. [2]. A mixture of bleomycin A2 and B2, marketed as After the onset of antibiotic synthesis, the supply of Blenoxane®, is administered by injection in clinic to treat limiting precursors is another key factor for improv- tumors, notably germ cell tumors, head and neck can- ing antibiotic yield [18]. Mannosylated glycans are com- cer and cervical carcinoma [3]. Although fermentation mon precursors of various glycoproteins and antibiotics medium optimization and manipulation of the pathway- including cell envelope polymers, polyenes, bleomycins specifc repressor blmR have been suggested to be use- and mannopeptimycins. Mannose can be sequentially ful for enhancing bleomycin production [4–6], the lower converted by phosphomannose isomerase (ManA), phos- yields and complex nutrient demands in rich medium phomannomutase (ManB), and GDP-mannose pyroph- have been major difculties in the industrial production osphorylase to form GDP-mannose. During bleomycin of bleomycins. biosynthesis, GDP-mannose and its isomer GDP-gulose Streptomyces species inhabit specifc environments are attached to the bleomycin aglycone through glyco- with changing nutrient availability, and their morpholog- sylation, which is responsible for the cellular recogni- ical development is tuned to coincide with the synthesis tion, uptake and metal ions chelation of bleomycins [19]. of secondary metabolites, including antibiotics. Under As the fnal step in the biosynthesis of most glycopep- starvation, multiple regulatory networks are employed by tide antibiotics, the genetic engineering of glycosylation Streptomyces to make a decision whether to continue veg- could not only result in hyper-susceptibility to antibiot- etative growth or form spores. Once it enters the spores ics and diverse derivatives of natural products, but its development, a series of genes are transcribed, which reaction efciency usually dictates the ultimate yield of activate the synthesis of antibiotics and morphological an antibiotic [20–23]. In addition to the glycosyl moie- diferentiation [7, 8]. Te onset of antibiotic synthesis not ties, the hybrid peptide-polyketide skeleton of bleomy- only depends on the expression of the specifc biosyn- cin is directly synthesized by nine common amino acids thetic gene clusters, but is also afected by the regulation [24], which indicate that the intracellular primary meta- of intracellular response mechanism of environmental bolic pathways of S. verticillus might also play a dominant status [9]. GlcNAc, the main component of chitin, is con- role in bleomycin biosynthesis. To further increase the sidered to be a signaling molecule with diferent efects yield of bleomycin, it is necessary to fully understand the on spore development and antibiotic yields in Streptomy- changes of global intracellular metabolites and identify ces under nutrient-rich/poor conditions [10, 11]. Actu- key modules that are associated with the synthesis of ble- ally, the regulation of the GntR/HutC family repressor omycins. Metabolic profling analysis is widely employed DasR has been found to be essential for the development for the identifcation of biomarkers and modules asso- and morphogenesis process, which controls the expres- ciated with target product synthesis. Tis approach has sion of genes involved in chitin/GlcNAc metabolism, the been used to increase the outputs of natural products development-related phosphoglucosyltransferase system and bioenergy [25, 26]. (PTS), stress response and nearly all the known antibiotic In this study, the signaling molecule GlcNAc greatly biosynthesis pathways [10, 12–15]. In vivo, DasR binds initiated the bleomycin biosynthesis in liquid medium. to a conserved element in the promoter region of its tar- To elucidate the GlcNAc induction mechanism, the get genes and represses their expression. However, the efects of the global transcription factor DasR on Glc- metabolites N-acetylglucosamine 6-phosphate (GlcNAc- NAc metabolism and bleomycin synthesis were investi- 6P) and glucosamine-6-phosphate (GlcN-6P) in GlcNAc gated by RT-qPCR and EMSAs. Te elevated expression catabolism would competitively bind to DasR, which is of the blmT gene under GlcNAc induction was found then released it from the target sites [14–16]. It has been to be the main cause of the improvement of bleomy- reported that the addition of GlcNAc to a niche simu- cin yield. To further increase bleomycin production lates a signal similar to cell wall lysis under nutritional under GlcNAc induction, gene blmT was overexpressed Chen et al. Microb

Load more